Arginyl-glutamine dipeptide for treatment of pathological vascular proliferation

ABSTRACT

The subject invention provides dipeptides useful in preventing pathological proliferation of blood vessels. The dipeptides of the subject invention are particularly advantageous because they are stable, bioavailable, and can be formulated in an aqueous solution, and in particular, into infant formulations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/506,413, filed Sep. 26, 2003.

BACKGROUND OF THE INVENTION

People suffering from visual impairment face many challenges inperforming routine daily activities and/or may not be able to fullyenjoy the visual aspects of their surroundings. Of particular concernwith regard to the current invention are visual impairments caused bydamage to the retina, which occur in conditions such as diabeticretinopathy and retinopathy of prematurity.

Diabetic retinopathy is a progressive disease characterized byabnormalities of the blood vessels of the retina caused by diabetes,such as weakening of the blood vessel walls, leakage from the bloodvessels, and bleeding and scarring around new vessels. Diabeticretinopathy results in impairment of a person's vision causing severelyblurred vision and, potentially, blindness.

Diabetes affects over 16 million Americans. The World HealthOrganization indicates that diabetes afflicts 120 million peopleworldwide, and estimates that this number will increase to 300 millionby the year 2025. Diabetics are faced with numerous complicationsincluding kidney failure, non-traumatic amputations, an increase in theincidence of heart attack or stroke, nerve damage, and loss of vision.Diabetic retinopathy is a form of visual impairment often suffered bydiabetics.

Due to significant medical advancements, diabetics are able to live muchlonger than in the past. However, the longer a person has diabetes thegreater the chances of developing diabetic retinopathy. Affecting over5.3 million Americans, diabetic retinopathy is the leading cause ofblindness among adults in the United States. Annually, in the UnitedStates, between 12,000 and 24,000 people lose their sight because ofdiabetes.

While management of diabetic retinopathy has improved, risk ofcomplications, such as loss of visual acuity, loss of night vision andloss of peripheral vision, remains significant and treatment sometimesfails. Currently, laser photocoagulation is the most effective form oftherapy for advanced disease. Unfortunately, current treatment optionsare inadequate and the disease is often progressive even with successfulglucose control.

Retinopathy of prematurity (ROP) is a disorder of retinal blood vesseldevelopment in the premature infant. Under normal development, bloodvessels grow from the back central part of the eye out toward the edges.In premature babies, this process is not complete and the abnormalgrowth of the vessels proliferate leading to scar tissue development,retinal detachment and possibly complete blindness.

ROP is the major cause of blindness in children under the age of 7. Thesalient pathological features are neovascularization in the retinalvascular endothelium with edema and breakdown in the blood-retinalbarrier (BRB) that leads to hemorrhage, tissue damage and retinalscarring ultimately leads, in the severest cases, to blindness.

Improved care in the neonatal intensive care unit has reduced theincidence of retinopathy of prematurity in moderately premature infants.Ironically, however, increasing rates of survival of very prematureinfants, who would have had little chance of survival in the past, hasincreased the occurrence of retinopathy of prematurity. Since these verypremature infants are at the highest risk of developing ROP, it is ofgreat concern that the condition may actually be becoming more prevalentagain.

For those babies in whom retinopathy progresses, treatment is necessary.Cryotherapy and laser treatment have some effect in advanced stages ofthe disease, saving a degree of vision in a proportion of the eyes thatwould otherwise have been blinded, but prevention awaits a betterunderstanding of major causative factors and underlying pathophysiology.

Current research shows promise that the prevention of retinal bloodvessel damage, which marks retinopathy, may be achieved by theutilization of certain compounds. It has been demonstrated that, inretinal epithelial cells, glutamine deprivation can lead to upregulationof vascular endothelial growth factor (VEGF) expression (Abcouwer S. etal., “Response of VEGF expression to amino acid deprivation and inducersof endoplasmic reticulum stress”, Invest Ophthalmol Vis Sci, August2002, pp. 2791-8, Vol. 43, No. 8). Most sick premature infants aredeprived of glutamine during the time they receive supplemental oxygen,a known predisposing factor in the development of ROP. The overexpression of VEGF during this time period is also thought to be involedin the pathogenesis of ROP providing glutamine supplements during thistime period could potentially down-regulate VEGF. Arginine is substratefor the reaction that produces nitric oxide, a very potent vasodilator,vasodilation in retinal blood vessels also prevents neovascularization.Nitirc oxide also has numerous other beneficial effects and is nowcommonly used for treatment of lung disease in critically ill infants.

It is well known that proteins are converted to amino acids in thedigestive system and that the resulting amino acids are used by the bodyfor growth and development. Proteins and peptides administered fortherapeutic or preventative measures are also well-known. Oligopeptidesare better absorbed in the intestines than individual amino acids.

European Patent Application No. 0,182,356 discloses a nutritionalcomposition containing at least one oligopeptide consisting of adipeptide or a tripeptide wherein the N-terminal amino acid residue isselected from the class consisting of alanine, lysine and arginine.

One group conducting research in this area concluded that glycine isgenerally superior to other amino acids as the N-terminal amino acidresidue in a dipeptide. This superiority was attributed to a greaterfraction of such an intravenously administered dipeptide reaches thetissues. S. Adibi et al., Influence of Molecular Structure on Half-lifeand Hydrolysis of Dipeptides in Plasma: Importance of Glycine asN-Terminal Amino Acid Residue, 35 Metabolism 850, 835 (1986).

Two European patents, 0,087,751 and 0,087,750 disclose water-solublepeptides. The '751 patent discloses a method to parenterally administerlow water-soluble amino acids. Two amino acids, tyrosine and cystine,individually have low solubility in water. These amino acids, however,are clinically useful and, therefore, it was desirable to find aneffective formulation. The '751 patent describes an infusion methodwhich involves bonding these relatively insoluble amino acids to theamino acid lysine to produce a tripeptide.

The '750 patent discloses the infusion of glutamine as a derivativesubstituted by α-aminoacyl residues on the a amino group. That is,glutamine is in the “c-terminal” position, in that its alpha aminonitrogen becomes part of the peptide bond with the other amino acid. Thepreferred dipeptide preparation disclosed in the '750 patent isalanyl-glutamine. The aminoacylation of glutamine is reported to achievea stabilization of the terminal amide group.

Experiments involving the use of total parenteral nutrition (TPN)containing glycyl-glutamine dipeptides, however, suggest potentialadverse effects of the TPN formulation containing glycyl-glutamine (U.S.Pat. No. 5,189,016).

Also, the use of an arginyl-glutamine dipeptide for the prevention ofmuscle breakdown and microbial infections has recently been described.See, WO 03/017787. These amino acids have also been described in complexcompositions (Miyazawa et al. (1976) Journal of Faculty of Fisheries andAnimal Husbandry Hiroshima 15(2):161-169; and JP 2119762).

Two commercially available dipeptides of glutamine are Dipeptiven, whichis an alanyl-glutamine (Fresenius Laboratories, Germany) and Glamin(Pharmacia and Upjohn Laboratory, Sweden), which is an amino acidsolution containing glycyl-glutamine dipeptide. To this date, there areno published studies of the arginyl-L-glutamine dipeptide.

With the increase of adult onset diabetes, longer life span fordiabetics and high rate of survival of very premature infants, manyindividuals are now at even greater risk for developing retinopathy.Although treatment options, such as laser therapy, exist for bothconditions, the results are inadequate and the disease often remainsprogressive. There remains a great need in the art for compositions andmethods which prevent and/or treat retinal diseases.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides materials and methods useful inpreventing proliferation of abnormal blood vessels. The prevention ofthe over-proliferation of these blood vessels according to the subjectinvention is particularly advantageous for treatment of certain ocularconditions including premature infants at risk for retinopathy ofprematurity and individuals at risk for diabetic retinopathy.

In a preferred embodiment of the subject invention, the amino acidsarginine and glutamine are combined as the dipeptide arginyl-glutaminein order to provide beneficial effects in a safe, easily absorbableformulation. The dipeptide of the subject invention is particularlyadvantageous because the solubility of the dipeptide is greater than theindividual amino acids.

Advantageously, the dipeptide of the subject invention inhibits theover-proliferation of unwanted blood vessels. The dipeptide of thesubject invention is also advantageous because it is safe for human andanimal use and can be readily formulated in an aqueous solution.

The compounds of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Ingeneral, the compositions of the subject invention will be formulatedsuch that an effective amount of the bioactive compound(s) is combinedwith a suitable carrier in order to facilitate effective administrationof the composition.

In accordance with the invention, pharmaceutical compositionscomprising, as an active ingredient, an effective amount of one or moreof the compounds and one or more non-toxic, pharmaceutically acceptablecarriers or diluents. Pharmaceutical carriers or excipients may containinert ingredients which do not interact with the compound, oringredients that do interact with the compound but not in a fashion soas to interfere with the desired effect. In general, the formulationsare prepared by uniformly and intimately bringing into association theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product. Examples of suchcarriers for use in the invention include ethanol, dimethyl sulfoxide,glycerol, silica, alumina, starch, and equivalent carriers and diluents.

The dipeptide of the subject invention can also be formulated as anutraceutical, including drinks, drink mixes, and bars.

BRIEF SUMMARY OF THE FIGURES

FIG. 1 shows inhibition of neovascularization by an arginyl-glutaminedipeptide in a model of retinopathy of prematurity; and

FIG. 2 shows inhibition of neovascularization by an arginyl-glutaminedipeptide in a model of retinopathy of prematurity at variousconcentrations of the dipeptide.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides compositions containing therapeuticdipeptides and methods for administering the same. The subject inventionprovides a novel, safe and affordable therapy for treatment ofpathological ocular vascular proliferation. Advantageously, the subjectinvention provides a dipeptide having excellent water solubility,stability to sterilization, long-term stability, and bioavailability forhumans and animals. One embodiment of the present invention provides acomposition comprising an aqueous pharmaceutical solution having atleast one arginyl-glutamine dipeptide. Other embodiments includenutraceutical formulations.

In a specific embodiment of the subject invention the arginyl-glutaminedipeptides described herein can be used for preventing the proliferationof abnormal retinal blood vessels in a patient. Thus, these dipeptidescan be administered to premature infants or diabetics who are at riskfor retinal disease.

In accordance with the teachings provided herein, aqueous clinicalcompositions can be prepared that include at least one arginyl-glutaminedipeptide. The dipeptide can be added to enteral or parenteralformulations. Each dipeptide has an N-terminal amino acid which isarginine. The C-terminal amino acid is glutamine.

The concentration of the dipeptide in the aqueous solution can be, forexample, from about 0.1 to about 25.0 percent by weight. As discussed inmore detail below, in addition to the dipeptides of the subjectinvention, the clinical solution can contain, for example, dextrose,liquid emulsions, vitamins, minerals and trace elements. The selectionof the particular dipeptide formulation depends upon the particular use.

For parenteral administration, a supply of the dipeptide solution may bemerged through a Y-connection with a supply of glucose solution or otherparenteral solutions. The dipeptide solutions may also be mixed withglucose solutions and/or other parenteral solutions to create a mixturewhich may be administered parenterally.

The administration of dipeptides rather than free amino acids allowsadministration of the same amount of amino acid residue in solutionswhich are less hypertonic and therefore can be introduced intoperipheral veins.

The dipeptides of the subject invention can be readily synthesizedand/or formulated by a person skilled in the art having the benefit ofthe instant disclosure. Alternatively, the dipeptides can be purchasedcommercially from, for example, Bachem Biosciences, Inc. which sells theH-Arg-Glu-OH salt.

The subject invention contemplates the administration of the Arg-Gludipeptide in any appropriate formulation including, for example, salts,prodrugs, and extended release formulations (such as, for example,formulation with polyethylene glycol (PEG)). The peptide itself may beadministered, as well as oligopeptides, peptides, proteins, proteinhydrolyzates, and any other materials that could serve as a source ofthe dipeptide.

In the case of oligopeptides, peptides, and proteins, these prodrugformulations may be designed with, for example, cleavage sites adjacentto each side of the dipeptide so that the dipeptide is generated uponexposure to enzymes, acids or other factors. In one embodiment, apolypeptide can be prepared with multiple dipeptides separated bycleavage sites, thereby creating multiple dipeptides upon exposure tothe cleaving factor. This cleaving to create the dipeptide can be doneas part of a production process or in vivo as the result of, forexample, digestive enzymes and/or acids. The source of the dipeptide maybe natural or the dipeptide (including prodrugs) may be producedrecombinantly or synthetically.

In one method, the subject invention involves identifying an individualwho has, or who is at risk for developing pathological vascularizationand then providing that individual with a composition comprising thedipeptide of the subject invention (or a prodrug thereof) along withinstructions or information concerning the activity of the dipeptide toinhibit pathological vascularization. In one embodiment, a nutriceuticalis provided that has the dipeptide, and/or a prodrug thereof, along withinstructions for consuming the nutriceutical to promote cardiovascular(or just vascular) health.

The compounds of the invention are useful for various therapeuticpurposes. Specifically, as described herein, the compounds of theinvention are effective for inhibiting vascular retinopathy and otherforms of pathological vascular proliferation. Accordingly, thesecompounds are useful prophylactically and therapeutically for treatinganimals and humans at risk for pathological vascular proliferationincluding vascular retinopathy and vasculature associated with tumors.

Therapeutic application of the compounds and compositions containingthem can be accomplished by any suitable therapeutic method andtechnique presently or prospectively known to those skilled in the art.

The peptides provided by the present invention are typicallyadministered to a mammal, particularly a human, dog or cat, any of whichis intended to be encompassed by the term “patient” herein, in need ofthe prevention or treatment of pathological vascular proliferation.Pathological conditions involving vascular proliferation include, forexample, tumor growth, age-related macular degeneration, vascularproliferation associated with angioplasty and/or stents, diabeticretinopathy and retinopathy of prematurity. Thus, the dipeptides of thesubject invention can be used to treat angiogenic diseases. Angiogenicdiseases include those that are disclosed in U.S. Pat. No. 5,759,547,which is incorporated herein, in its entirety, by reference.

Angiogenesis and neovascularization in the adult animal is usually apathological process, and is in direct contradistinction tonon-pathological neovascularization, which usually occurs in normalembryogenesis (e.g., development of the embryonic vascular system). Inaccordance with the subject invention, neovascularization refersspecifically to pathological neovascularization. Aberrant orpathological vascularization is a key component in numerous diseasestates. For example, vascularization is a critical element of most solidtumors, such as cancers of the brain, genitourinary tract, lymphaticsystem, stomach, larynx and lung. These include histiocytic lymphoma,lung adenocarcinoma and small cell lung cancers. Aberrant vasculargrowth in the retina can lead to visual degeneration which can culminatein blindness. Accordingly, the subject invention provides dipeptidecompounds and formulations thereof for the treatment ofneovascularization.

Compounds of the invention can also be used to inhibit the proliferationof vascular endothelial cells and so are indicated for use in treatinggraft vessel diseases such as restenosis or vascular occlusion followingvascular insult such as angioplasty, allo- or xenotransplantvasculopathies, graft vessel atherosclerosis, and in the transplantationof an organ (e.g., heart, liver, lung, kidney or pancreatic transplants(Weckbecker et al., Transplantation Proceedings 1997, 29, 2599-2600).

The peptides are administered by incorporating the peptide in apharmaceutical composition comprising the peptide or a non-toxicpharmaceutically acceptable salt or prodrug thereof and a non-toxicpharmaceutically acceptable carrier therefor.

The peptide or its salt or prodrug is employed in an effective amounti.e. an amount sufficient to evoke the desired pharmacological response.This is generally an amount sufficient to produce lessening of one ormore of the effects of pathological vascular proliferation. In the caseof retinopathy, it is an amount sufficient to produce regression ofneovascularization and/or an amount sufficient to produce improvedvisual acuity.

The terms “pharmaceutically acceptable carrier” or a “carrier” refer toany generally acceptable excipient or drug delivery device that isrelatively inert and non-toxic. The peptide can be administered with orwithout a carrier. When treating retinopathies, a preferred embodimentis to administer the peptide to the retinal area or the vasculaturearound or leading to the retina. Exemplary carriers include calciumcarbonate, sucrose, dextrose, mannose, albumin, starch, cellulose,silica gel, polyethylene glycol (PEG), dried skim milk, rice flour,magnesium stearate, and the like. The peptide can be administeredsystemically or locally (e.g., by injection or diffusion). Suitablecarriers (e.g., pharmaceutical carriers) also include, but are notlimited to sterile water, salt solutions (such as Ringer's solution),alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc. Such preparations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likewhich do not deleteriously react with the active compounds. They canalso be combined where desired with other active substances, e.g.,enzyme inhibitors, to reduce metabolic degradation. A carrier (e.g., apharmaceutically acceptable carrier) is preferred, but not necessary toadminister the peptide.

Suitable non-toxic pharmaceutically acceptable carriers for use with thepeptide will be apparent to those skilled in the art of pharmaceuticalformulation. See, for example, Remington's Pharmaceutical Sciences,seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company,Easton, Pa. (1985). The choice of suitable carriers will depend upon theexact nature of the particular dosage form selected.

The supplement can take on various forms, including but not limited topills, edible bars, drinks or drink mix. The compounds of the subjectinvention may be combined with other components such as, for example, asoluble fiber compound. The soluble fiber compound may be, for example,locust gum, guar gum, pectin, gum arabic, or psyllium.

The person skilled in this art, having the benefit of the currentdisclosure can readily formulate the compounds of the subject inventioninto a pill, bar, or other edible composition for easy and enjoyableconsumption. These therapeutic compositions can be used as describedherein.

In one embodiment, the present invention provides a nutriceuticalcomposition. The dipeptides can be obtained or produced using processesknown to those skilled in the art having the benefit of the instantdisclosure.

The compositions of the invention are useful for various therapeuticpurposes. Because of the antiproliferative properties of the compounds,they are useful to prevent unwanted cell growth in a wide variety ofsettings including in vitro uses.

Therapeutic application of the new compositions can be accomplished byany suitable therapeutic method and technique presently or prospectivelyknown to those skilled in the art.

When used to reduce the severity or incidence of tumors, the dosageadministration to a host in the above indications will be dependent uponthe identity of the cancer cells, the type of host involved, its age,weight, health, kind of concurrent treatment, if any, frequency oftreatment, and therapeutic ratio.

In a preferred embodiment, the active ingredient at the site oftreatment is the dipeptide of the subject invention in the absence ofother amino acids. Thus, it is not necessary for the subject inventionto be specifically formulated with, for example, an essential amino acidand a non-essential amino acid.

In one embodiment, the dipeptide of the subject invention can beadministered as a nutriceutical supplement in unit dosage form. In suchform, the preparation is subdivided into unit doses containingappropriate quantities of the nutriceutically active component. The unitdosage form can be a packaged preparation, such as packeted tablets,capsules, lotions, ointments and powders in vials or ampoules. Also, theunit dosage can be a drink (such as a powder-based drink, shake or teaformulation), yogurt, solid food product, capsule, tablet, lotion,ointment, chewing gum, lozenge, or it can be the appropriate number ofany of these in packaged form.

In one embodiment, there is provided a nutriceutical of the presentinvention in the form of a food bar. The food bar, which may be cookedor non-cooked, may contain a grain or grains, nuts, possibly driedfruit, sweeteners and other ingredients that may be mixed with a binder,such as a sugar syrup or shortening, and formed into “bars” of desirablesize. Generally, food bars are prepared in bar form of a manageable sizefor a product of this nature, and bearing in mind both package size andtime required for consumption, weigh in the range of 2-3 ounces.

The food bar of the present invention may contain an amount of proteinin addition to significant amounts of complex and simple carbohydrate,such as those non-cooked food bars described by U.S. Pat. No. 4,055,669.Further, the food bar of the present invention may contain dietary fiberto aid in the normalization of bowel function and reduce the risk ofcolonic diseases. There are presently available a number of non-cookedfood bars that provide varying amounts of dietary fiber while possessingrequisite qualities of acceptable taste and texture, including food barsdescribed in U.S. Pat. Nos. 4,673,578 and 4,871,557.

The therapeutic dosage range can be determined by one skilled in the arthaving the benefit of the current disclosure. Naturally, suchtherapeutic dosage ranges will vary with the size, species and physicalcondition of the patient, the severity of the patient's medicalcondition, the particular dosage form employed, the route ofadministration and the like. In addition, a route of administration maybe selected to slowly release the chemical, e.g., slow intravenousinfusion. The inventive composition that contains the subject dipeptidemay be a nutritional composition (nutritionally complete or nutritionalsupplement) for enteral administration. That is, it is designed fororal, intragastric, or transpyloric use.

The composition of the invention may be an infant formula or adultnutritional composition that can be milk-based, soy-based, or based onother food sources. The composition may be prepared as a powder orliquid nutritional composition for formulas prepared for infant,pediatric and adult populations. The inventive composition may beprepared as a nutritionally complete diet by including vitamins andminerals at acceptable levels. The subject composition can be in theform of a dietary product such as an infant formula, milk substitute,and meal replacement or supplement.

An embodiment of the invention is a dietary supplement that containsarginyl-glutamine dipeptide, or a precursor thereof (which may also bereferred to herein as a prodrug). The dietary supplement is designed tobe administered along with a food or nutritional composition, such asinfant formula, and can either be intermixed with the food ornutritional composition prior to ingestion by the subject, or can beadministered to the subject either before or after ingestion of a foodor nutritional composition. The subject dietary supplement contains anamount of arginyl-glutamine dipeptide, or a precursor thereof, that iseffective for the prevention or treatment of retionoathy of prematurity,diabetic retinopathy, vascular proliferative retinopathy, orproliferation of abnormal vascularization, and the like.

In one embodiment, a novel infant formula containing arginyl-glutaminedipeptide, or precursor thereof, is nutritionally complete. By the term“nutritionally complete” is meant that the composition contains adequatenutrients to sustain healthy human life for extended periods. The infantformula of the invention contains ingredients which are designed to meetthe nutritional needs of the human infant namely, a protein,carbohydrate and lipid source and other nutrients such as vitamins andminerals.

Besides the subject dipeptide, the composition of the invention containsa nitrogen source (i.e., amino acids and/or protein) in an amount thatis typically about 1 g to about 10 g per 100 kcal of total composition,preferably about 2 g to about 6 g per 100 kcal; the amount of lipidsource per 100 kcal of total composition is typically greater than 0 gup to about 6 g, preferably about 0.5 g to about 5.5 g and morepreferably about 2 g to about 5.5 g; and the amount of non-fibercarbohydrate source per 100 kcal of total composition is typically about5 g to about 20 g, preferably about 7.5 g to about 15 g. The amount ofvitamins and minerals in the nutritionally complete composition istypically sufficient to meet 100% of the U.S. recommended daily intake(RDI) in about 500 to about 3,000 kcal, preferable is about 1,000 toabout 3,000 kcal.

In one embodiment of the present nutritional composition the amount ofvitamins and minerals is sufficient to meet 100% of the RDI in about 500to about 3,000 kcal, preferably in about 1,000 to about 3,000 kcal. Asused herein, the RDI's are intended to mean those published in theFederal Register, Vol. 58, No. 3, Wednesday, Jan. 6, 1993, page 2227which are as follows: Vitamin A, 5,000 International Units; Vitamin C,60 milligrams; Thiamin, 1.5 milligrams; Riboflavin, 1.7 milligrams;Niacin, 20 milligrams; Calcium, 1.0 gram; Iron, 18 milligrams; VitaminD, 400 International Units; Vitamin E, 30 International Units; VitaminB₆, 2.0 milligrams; Folic acid, 0.4 milligrams; Vitamin B₁₂, 6micrograms; Phosphorus, 1.0 gram; Iodine, 150 micrograms; Magnesium, 400milligrams; Zinc, 15 milligrams; Copper, 2 milligrams; Biotin, 0.3milligram; Pantothenic acid, 10 milligrams.

In one embodiment, the novel infant formula contains thearginyl-glutamine dipeptide, or a precursor thereof, in an amount thatis less than 0.1% by weight of the formula. It is preferred that theamount of arginyl-glutamine dipeptide in the formula is from about0.001% to 0.098% by weight of the formula, more preferred is an amountof from about 0.01% to 0.098% by weight.

In the present method, the subject infant formula or dietary supplementis administered to an infant in an amount that is sufficient to preventor treat retionoathy of prematurity, diabetic retinopathy, vascularproliferative retinopathy, or proliferation of abnormal vascularization.In a preferred embodiment, that amount is from about 0.001 to about10,000 mg/kg of body weight of the subject per day, more preferred is anamount of from about 0.001 to about 100 mg/kg/day, yet more preferred isabout 0.01 to about 50 mg/kg/day, and even more preferred is about 0.01to about 10 mg/kg/day, and yet more preferred is about 0.01 to about 1mg/kg/day. Alternatively, the amount administered to an infant is fromabout 1 mg to about 10,000 mg/day, preferably abut 10 mg to about 1000mg, and yet more preferred about 10 mg to about 500 mg.

The protein source that is present in addition to the subject dipeptidecan be non-fat milk solids, a combination of non-fat milk solids andwhey protein, a partial hydrolysate of non-fat milk and/or whey solids,soy protein isolates, or partially hydrolyzed soy protein isolates. Theinfant formula can be casein predominant or whey predominant.

The carbohydrate source in the infant formula can be any suitablecarbohydrate known in the art to be suitable for use in infant formulas.Typical carbohydrate sources include sucrose, fructose, glucose,maltodextrin, lactose, corn syrup, corn syrup solids, rice syrup solids,rice starch, modified corn starch, modified tapioca starch, rice flour,soy flour, and the like.

The lipid source in the infant formula can be any lipid or fat known inthe art to be suitable for use in infant formulas. Typical lipid sourcesinclude milk fat, safflower oil, egg yolk lipid, olive oil, coconut oil,palm oil, palm kernel oil, soybean oil, sunflower oil, fish oil andfractions derived thereof such as palm olein, medium chain triglycerides(MCT), and esters of fatty acids wherein the fatty acids are, forexample, arachidonic acid, linoleic acid, palmitic acid, stearic acid,docosahexaenoic acid, eicosapentaenoic acid, linolenic acid, oleic acid,lauric acid, capric acid, caprylic acid, caproic acid, and the like.High oleic forms of various oils are also contemplated to be usefulherein such as high oleic sunflower oil and high oleic safflower oil.Medium chain triglycerides contain higher concentrations of caprylic andcapric acid than typically found in conventional oils, e.g.,approximately three-fourths of the total fatty acid content is caprylicacid and one-fourth is capric acid.

Nutritionally complete compositions contain all vitamins and mineralsunderstood to be essential in the daily diet and these should be presentin nutritionally significant amounts. Those skilled in the artappreciate that minimum requirements have been established for certainvitamins and minerals that are known to be necessary for normalphysiological function. Practitioners also understand that appropriateadditional amounts (overages) of vitamin and mineral ingredients need tobe provided to compensate for some loss during processing and storage ofsuch compositions.

To select a specific vitamin or mineral compound to be used in theinfant formula of the invention requires consideration of thatcompound's chemical nature regarding compatibility with the particularprocessing conditions used and shelf storage.

Examples of minerals, vitamins and other nutrients optionally present inthe composition of the invention include vitamin A, vitamin B₆, vitaminB₁₂, vitamin E, vitamin K, vitamin C, folic acid, thiamine, inositol,riboflavin, niacin, biotin, pantothenic acid, choline, calcium,phosphorus, iodine, iron, magnesium, copper, zinc, manganese, chloride,potassium, sodium, selenium, chromium, molybdenum, taurine, andL-carnitine. Minerals are usually added in salt form. In addition tocompatibility and stability considerations, the presence and amounts ofspecific minerals and other vitamins will vary somewhat depending on theintended infant population.

The infant formula of the invention also typically contains emulsifiersand stabilizers such as soy lecithin, carrageenan, and the like.

The infant formula of the invention may optionally contain othersubstances which may have a beneficial effect such as lactoferrin,nucleotides, nucleosides, immunoglobulins, and the like.

The infant formula of the invention is in concentrate liquid form,liquid ready to consume form, or powder form. Of course, if in powderform, the formula is diluted to normal strength with water to be in aform ready to consume.

The osmolality of the liquid infant formula of the invention (when readyto consume) is typically about 100 to 1100 mOsm/kg H₂O, more typicallyabout 200 to 700 mOsm/kg H₂O.

The infant formula of the invention can be sterilized, if desired, bytechniques known in the art, for example, heat treatment such asautoclaving or retorting, and the like.

The infant formula of the invention can be packaged in any type ofcontainer known in the art to be used for storing nutritional productssuch as glass, lined paperboard, plastic, coated metal cans and thelike.

The infant formula of the invention is shelf stable afterreconstitution. By “shelf stable” is meant that the formula in a formready to consume remains in a single homogenous phase (i.e., does notseparate into more than one phase upon visual inspection) or that thethickener does not settle out as a sediment upon visual inspection afterstorage overnight in the refrigerator. With the thickened nature of theproduct, the formula of the invention also has the advantage ofremaining fluid (i.e., does not gel into a solid mass when storedovernight in the refrigerator).

In the method of the invention, infant formula comprisingarginyl-glutamine dipeptide, or a precursor thereof, is administered toan infant. The form of administration is oral, which includes tubefeeding.

The invention provides a commercially acceptable product in terms ofdesired stability and physical characteristics and the productdemonstrates little to no observable browning effect by-productsassociated with a Maillard reaction.

Further, the inventive composition is substantially homogeneous for anacceptable period after reconstitution (or for the shelf-life ifprepared as a liquid). The invention is particularly useful for infantformula preparations for the prevention and treatment of retionoathy ofprematurity, although it is equally applicable to other elemental dietsspecific to a selected population that is at risk of, or is suspected ofhaving, diabetic retinopathy, vascular proliferative retinopathy, orproliferation of abnormal vascularization, and the like.

One embodiment of the current invention envisions parenteraladministration, especially intravenous administration, as the route ofadministration. Parenteral dosage forms should be sterile andpyrogen-free, and are prepared in accord with accepted pharmaceuticalprocedures. The parenteral formulations may be organic or aqueous ormixed organic/aqueous formulations and may further containanti-oxidants, buffers, bacteriostats, isotonicity adjusters and likeadditions acceptable for parenteral formulations. In a preferredembodiment, the parenteral formulation contains an effective amount ofthe peptide of the subject ivention in an aqueous solution.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants. In particular, carriers forparenteral administration include aqueous solutions of dextrose, saline,pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,polyoxyethylene-polyoxypropylene block polymers, and the like. Ampulesare convenient unit dosages.

Also, according to the subject invention, the local administration ofthe dipeptide compounds, and formulations thereof, by means of a drugdelivery device or implant placed in proximity to the local tissue siteprovides for the maintenance of efficacious, safe levels of active drugingredient at the local tissue disease site.

According to the subject invention, the local ocular administration ofdipeptide compounds of the invention, and/or formulations thereof,attenuate ocular pathological disease processes. Thus, local ocularadministration of a dipeptide compound of the invention, and/orformulations thereof, provides for an efficacious but safe controlledconcentration range of the dipeptide directly in the eye.

Ocular dipeptide-based therapies, as describe herein, providesignificant advantages for treating neovascular ocular disease relativeto current laser surgery treatment modalities including panretinalphotocoagulation, which can be accompanied by extensive ocular tissuedamage. In the examples of posterior neovascular ocular diseases, suchas Age Related Macular Degeneration and Diabetic Retinopathy, targetocular pathologies and tissues for treatment are especially localized tothe retinal, choroidal and corneal ocular compartments.

Preferably, the peptide is administered locally to the eye, retinalarea, choroid area or associated vasculature. The peptide can also beadministered to the cornea of the eye. The peptide diffuses into the eyeand contacts the retina or surrounding vasculature (e.g., eye drops,creams or gels).

The dipeptide compounds of the present invention, and formulationsthereof, are advantageous because they overcome problems associated withstability, toxicity, lack of target tissue specificity, safety,efficacy, extent and variability of bioavailability.

As contemplated in the subject invention, where a dipeptide compoundcomprises a prodrug, the prodrug can be converted to a biologicallyactive compound at a controlled rate via passive (such as by aqueoushydrolysis) or biologically mediated (such as biocatalytic or enzymatic)mechanisms. An advantage of the in vivo conversion of the prodrug isthat the ensuing dipeptide provides localized therapeutic effects intarget disease tissue with high therapeutic margins of safety.

A further embodiment provides the use of dipeptide compounds inconjunction with a drug delivery system in the form of an implant or adevice for the treatment of conditions as set forth herein. Certainembodiments of the invention contemplate the use of dipeptide compoundsand formulations thereof for use as coatings for example in conjunctionwith physical implants such as stents and band ligatures. Therapeuticuses of such implants include but are not limited to vascular diseasessuch as restenosis, and in bone and tissue grafts.

A further embodiment of the subject invention provides for the localadministration of dipeptide compounds in combination with otherpharmacological therapies. As contemplated in the subject invention,combination therapies of dipeptide compounds with other medicamentstargeting similar or distinct disease mechanisms have advantages ofgreater efficacy and safety relative to respective monotherapies witheither specific medicament.

In one embodiment, a dipeptide compound is used to treat neovascularocular disease by localized (for example, in ocular tissue) concurrentadministration with other medicaments that act to block angiogenesis bypharmacological mechanisms. Medicaments that can be concurrentlyadministered with a dipeptide compound of the invention include, but arenot limited to, vascular endothelial growth factor VEGF blockers (e.g.by VEGF neutralizing binding molecules such as Macugen (Eyetech) andLucentis (ranibizumab, Genentech), Squalamine lactate (GenaeraCorporation); and VEGF tyrosine kinase inhibition) for treatingneovascular ocular disease (AMD and Diabetic Retinopathy) andglucocorticoids (e.g. Triamcinolone) for treating macular edema.

One or more active agents can be administered. When administering morethan one, the administration of the agents can occur simultaneously orsequentially in time. The agents can be administered before and afterone another, or at the same time. The methods also includeco-administration with other drugs that are used to treat retinopathy orother diseases described herein.

The composition can be administered in a single dose or in more than onedose over a period of time to confer the desired effect.

The dosage administration to a host in the above indications will bedependent upon the specific condition being treated, the type of hostinvolved, its age, weight, health, kind of concurrent treatment, if any,frequency of treatment, and therapeutic ratio. Those skilled in the artwill be able to determine the appropriate dosages depending on these andother factors. Typically, a therapeutically effective amount of thecompound can range from about 1 mg per day to about 1000 mg per day foran adult human animal. For oral administration to human adults, a dosageof 0.01 to 100 mg/kg/day, preferably 0.01-1 mg/kg/day, is generally atherapeutically effective amount.

To provide for the administration of such dosages for the desiredtherapeutic treatment, new pharmaceutical compositions of the inventionwill advantageously comprise between about 0.1% and 45%, and especially,1 and 15%, by weight of the total of one or more of the new compoundsbased on the weight of the total composition including carrier ordiluent.

In a retinal cell culture model used to study the effects of thedipeptide on transepithelial resistance (TER) and vascular endothelialgrowth factor (VEGF), it was demonstrated that the dipeptide increasedTER and decreased VEGF, both desirable effects that have been associatedwith a decrease in vascular proliferative retinal disease.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following is an example which illustrates procedures for practicing theinvention. This example should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Use of Arginyl-glutamine to Prevent Retinopathy of Prematurity

Neonatal mice were exposed to a period of hyperoxia to induce retinalangiogenesis that mimics retinopathy of prematurity. AnArginyl-glutamine dipeptide was administered intraperitoneally twicedaily during the period when neovascularization occurs in this model,days 12 through 17.

On day 17 the animals are sacrificed and degree of angiogenesis wasquantified by counting pre-retinal neovascularization on stainedsections from treated and untreated animals.

The data summarized in FIG. 1 represent combined data from two separateexperiments. The results demonstrated a statistically significant effectof the dipeptide (argininyl-glutamine) compared to vehicle and dipeptide(alaninyl-glycine). Treatment of the pups with the dipeptide(argininyl-glutamine) resulted in a 80% reduction in preretinalneovascularization.

This experiment has been repeated with different doses of the dipeptidecompared to the vehicle. The results are shown in FIG. 2. This shows aclear decrease in nuclei (associated with decreased retinopathy) in theanimals receiving the dipeptide after the oxygen stress challenge thatis known to induce retinopathy.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. An infant formula comprising a source of arginyl-glutamine dipeptide.2. The infant formula according to claim 1, wherein the source ofarginyl-glutamine dipeptide is arginyl-glutamine dipeptide, or aprecursor thereof.
 3. The infant formula according to claim 1, whereinthe infant formula comprises arginyl-glutamine dipeptide, or a precursorthereof, in an amount sufficient for the prevention or treatment of acondition that is selected from retinopathy of prematurity, diabeticretinopathy, vascular proliferative retinopathy, or proliferation ofabnormal vascularization.
 4. The infant formula according to claim 1,wherein the infant formula is nutritionally complete.
 5. The infantformula according to claim 3, wherein the infant formula containsarginyl-glutamine dipeptide, or precursor thereof, in an amount lessthan 0.1% by weight.
 6. The infant formula according to claim 3, whichcontains an amount of arginyl-glutamine dipeptide of from about 0.001%to 0.098% by weight of the formula.
 7. The infant formula according toclaim 2, wherein the source of arginyl-glutamine dipeptide is one ormore of arginyl-glutamine containing oligopeptides, polypeptides,peptides, or proteins.
 8. A method for the prevention or treatment of acondition that is selected from retinopathy of prematurity, diabeticretinopathy, vascular proliferative retinopathy, or proliferation ofabnormal vascularization, in an infant, the method comprisingadministering to the infant an infant formula or dietary supplementcontaining arginyl-glutamine dipeptide, or precursor thereof, in anamount that is sufficient to treat or prevent the condition.
 9. Themethod according to claim 8, wherein administering comprisesadministering a nutritionally complete infant formula.
 10. The methodaccording to claim 8, wherein the infant is administered an amount ofthe formula or supplement sufficient to provide the infant thearginyl-glutamine dipeptide in an amount of from about 0.001 to about10,000 mg/kg of body weight of the infant per day.
 11. The methodaccording to claim 8, wherein infant is administered an amount of theformula or supplement sufficient to provide the infant thearginyl-glutamine dipeptide in an amount of from about 0.001 to about100 mg/kg of body weight of the infant per day.
 12. The method accordingto claim 8, wherein infant is administered an amount of the formula orsupplement sufficient to provide the infant the arginyl-glutaminedipeptide in an amount of from about 0.01 to about 1 mg/kg of bodyweight of the infant per day.
 13. The method according to claim 8,wherein the infant is a premature infant.
 14. The method according toclaim 8, wherein the infant is a newborn infant.